Biochemical and serological characteristics of natural 9-O-acetyl GD3 from human melanoma and bovine buttermilk and chemically O-acetylated GD3.
Because its expression appears to be largely restricted to human melanomas, 9-O-acetyl-GD3 is a candidate antigen for vaccine construction. Searching for potential sources, we compared chemically O-acetylated calf brain GD3 and 9-O-acetyl-GD3 extracted from bovine buttermilk with 9-O-acetyl-GD3 from human melanoma. Three fractions (F1-F3) of chemically O-acetylated GD3 differed in the number and position of O-acetyl groups. O-Acetylation sites were the lactose portion in F1 and lactose as well as sialic acid in F2 and F3. Natural (melanoma- or buttermilk-derived) 9-O-acetyl-GD3 was O-acetylated solely on the sialic acid moiety. While F1 was not reactive with monoclonal antibodies against 9-O-acetyl-GD3, F2 and F3 were as reactive as the natural products. Immunization with the natural products induced high-titer antibodies against natural 9-O-acetyl-GD3 as well as F2 and F3. In contrast, mice immunized with the synthetic fractions produced antibodies only against the immunogen but not against natural 9-O-acetyl-GD3. Only immunization with the natural products induced production of antibodies reactive with surface antigens of melanoma cells expressing 9-O-acetyl-GD3. The findings suggest (a) that C-9 of the subterminal sialic acid is the site of chemical O-acetylation in F2 and F3, as opposed to C-9 of the terminal sialic acid in the natural products; (b) that O-acetylation of both the terminal and subterminal sialic acid moieties of GD3 results in recognition by three murine monoclonal antibodies (D1.1, ME 311, and Jones) reactive with human melanoma cells; (c) that O-acetylation of the terminal sialic acid is critical, on the other hand, for inducing an immune response against melanoma 9-O-acetyl-GD3; and (d) that O-acetyl GD3 from bovine buttermilk can substitute as immunogen for inducing an immune response against human melanoma cell surface antigens in the mouse.
Ritter
G
G
Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Boosfeld
E
E
Markstein
E
E
Yu
R K
RK
Ren
S L
SL
Stallcup
W B
WB
Oettgen
H F
HF
Old
L J
LJ
Livingston
P O
PO
D000107:Acetylation; D000818:Animals; D002079:Butter / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005260:Female; D005732:Gangliosides / Q000032:analysis* / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007114:Immunization; D007074:Immunoglobulin G / Q000096:biosynthesis*; D008545:Melanoma / Q000032:analysis* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL
D005732:Gangliosides; D007074:Immunoglobulin G; C026226:ganglioside, GD3; D002079:Butter; C049132:9-O-acetyl-GD3 ganglioside